Faculty Collaboration Database

1H and 15N magnetic resonance assignments, secondary structure, and tertiary fold of Escherichia coli DnaJ(1-78). Biochemistry 1995 Apr 25;34(16):5587-96

Date

04/25/1995

Pubmed ID

7727420

DOI

10.1021/bi00016a033

Scopus ID

2-s2.0-0029651968 (requires institutional sign-in at Scopus site)   70 Citations

Abstract

We report the 1H and 15N chemical shift assignments along with an NMR-derived preliminary structure for DnaJ(1-78), a highly conserved N-terminal domain of DnaJ, the Escherichia coli Hsp40 homolog. This 9 kDa domain is believed to cooperate with DnaK, the E. coli Hsp70 homolog, in regulating a variety of cellular functions. Heteronuclear 3D NMR experiments were carried out on a uniformly 15N-labeled DnaJ(1-78), which is a stable, folded fragment. Standard 15N-edited NMR techniques afforded complete assignment of the backbone amide 1H and 15N pairs and partial assignment of the side-chain 1H and 15N atoms. The secondary structure of DnaJ(1-78) was determined from NOE connectivities obtained from 3D 15N-separated and 2D homonuclear NOESY spectra as well as 3JHNH alpha coupling constants obtained from a DQF-COSY spectrum and a 15N-edited HNHA experiment. The stability of secondary structural elements was assessed by monitoring amide exchange rates, and a model for the three-dimensional fold of these elements was derived from a set of long-range contacts extracted from homonuclear 2D NOESY experiments. The analysis indicates that DnaJ(1-78) is comprised of four alpha-helices and no beta-sheet with a short unstructured loop between antiparallel helices II and III. The shorter N-terminal and C-terminal helices make contacts with helices II and III at points well removed from the central loop. A discussion of how this preliminary structural model may explain mutation data from other laboratories is presented.

Author List

Hill RB, Flanagan JM, Prestegard JH

MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Bacterial Proteins
Base Sequence
Cloning, Molecular
DNA Primers
Escherichia coli
Escherichia coli Proteins
HSP40 Heat-Shock Proteins
Heat-Shock Proteins
Hydrogen
Magnetic Resonance Spectroscopy
Models, Structural
Molecular Sequence Data
Nitrogen Isotopes
Peptide Fragments
Polymerase Chain Reaction
Protein Structure, Secondary
Protein Structure, Tertiary
Recombinant Proteins